2-8 Whole-plant recovery ratings as a percent of control plants after 18 day
recovery in greenhouse after freeze treatment (-3.20C) while root system was
kept above freezing (50C) February 2002 .............................. 45

5-2 Crosses and self-pollinations made August 2003 through December 2003 of
freeze-tolerant, intermediate, and freeze-sensitive bahiagrass clones ........... 99

5-3 Percent emergence of crosses and self-pollinations made August 2003 through
December 2003 of freeze-tolerant, intermediate, and freeze-sensitive
bahiagrass clones .................................. .......... 100

5-6 Mean canopy leaf freeze-damage ratings (1 to 9) of progeny rated after a
freeze event (17 December 2004) as a result of female parent and pollen
source ............ ............................................. 102

5-7 Analysis of variance of canopy damage ratings made after 17 December 2004
freeze using the fixed model Griffing's Method 3, and 6 bahiagrass clones
varying in LTFT trait, and their progeny ............................. 104

Figure 1-2. Freeze-injury symptom response of plants leading to reversible or
irreversible cell and plant injury (Conceptualized by the author based on
data from Sakai and Larcher, 1987; Nilsen and Orcutt, 1996; Guy, 2003)

apoxic, stressing roots with a lack of oxygen, in addition to having foliage frozen.

Cumulative stress may have accounted for unexpectedly sensitive whole-plant freeze-

damage ratings.

Table 2-8. Whole-plant recovery ratings as a percent of control plants after 18 day
recovery in greenhouse after freeze treatment (-3.20C) while root system
was kept above freezing (50C) February 2002.

Absolute vessel diameter values of FL9 and FL67 were higher than those reported

(Table 3-1) for all temperate woody plants except for oak (250 rim) and within the range

of five of the tropical vining species (120 to 200 rim).

Simple mean leaf position effects were significant across both lines (Table 3-3).

The xylem diameter of the first fully expanded leaf was significantly smaller than the

second and third fully expanded leaves. The results show that what had been defined as

the first fully expanded leaf was still experiencing anatomical changes when sampled.

Table 3-3. Mean midrib xylem diameter of three leaf positions across two lines (FL9,
FL67) varying in LTFT, sampled 1 January 2003.
Leaf position Xylem diameter Number of measurements
gIm n
First 158c* 120
Second 190a 80
Third 184b 80
*Means with the same letter are not significantly different at P = 0.05 confidence level.

Observation in the various controlled freezer trials (Chapter 2) indicated that

freeze-injury symptoms appeared more readily on older leaves, such as the second and

third fully expanded leaves, rather than on the first fully expanded leaf and the new leaf

emerging from the whorl. Leaf position (thus leaf age) freezing sequence may be similar

in bahiagrass as in Hordeum sp. (barley) (Pearce and Fuller, 2001b), depending on the

severity of the freezing treatment. When organs of uprooted barley were tested under

controlled freeze conditions in the laboratory, freezing order occurred first from

nucleated leaves, roots, older leaves, and younger leaves with secondary tillers being the

56

last to freeze (Pearce and Fuller, 2001b). If the xylem diameter is one of the mechanisms

that is related to LTFT it might explain why older leaves freeze before younger leaves.

Visualization of the third emerged fully expanded leaf cross-section of the freeze-

3 Not reversible Reversible Chill stress 0 0 C Time 20 0 C Reversible Chill stress 0 0 C Time 20 Critical temperature a need to define some terms used in the literature and review basic mechanisms that apply to cold injury and plant cold-tolerance. Chill-injury is foliar, and whole-plant damage, as a result of temperatures above 0C and below some threshold temperature unique for that species and even for a specific genotype. Freeze-injury is plant damage at temperatures below 0C or when radiative frosts occur with ice formation (defined later). Chill-injury can be conceptualized as the plant responding with increasing damage to cold stress as ambient temperature approaches 0C, and depending on the duration of that stress (Figure 1-1). Damage to bahiagrass foliage from chill stress has not been documented. However, there may be mechanism continuums that may apply to further our understanding of freeze-injury in bahiagrass. Figure 1-1.Chill-injury symptom response of chill-sensitive plants leading to reversible or irreversible cell and plant injury (Adapted from Nilsen and Orcutt, 1996) Temperature ranges where damage to chill-sensitive plants occurs vary. Values such as from 12 to 0C (Buchanan et al., 2000.) and from to 20 to 0C (Lyons et al.,

8 0 C -10 C -3 C -50 C Freezesensitive Freezetolerant Heterogeneous nucleation Homogenous nucleation Dehydration tolerance + Membrane fatty acids + Cryoprotectants Heterogeneous nucleation after supercooling due to solute accumulation Time in peninsular Florida. Bahiagrass genotypes with freeze-tolerance may have more than one protective mechanism. Figure 1-2.Freeze-injury symptom response of plants leading to reversible or irreversible cell and plant injury (Conceptualized by the author based on data from Sakai and Larcher, 1987; Nilsen and Orcutt, 1996; Guy, 2003) One protective mechanism postulated (Levitt, 1978; Pearce, 2001) for shallow freezes (-1 to -3C) has been freezing-point depression of cell sap by heterogeneous ice nucleation after transient supercooling. Therefore, freeze-tolerant bahiagrass genotypes may be transiently protected by supercooling during short freeze events. Supercooling is simply defined as water or a solution below the equilibrium freezing point of water (0 to -1C) and above the homogeneous nucleation temperature of water (-40 to -41C) (Chen et al., 1995). Supercooling is a mechanism that allows plants to avoid what could be lethal intracellular freezing by reducing the freezing point of the cell solution (Hudak, J. and J. Salaj, 1999). Supercooling is an unstable thermodynamic situation when a liquid solution is not in phase equilibrium with the solid ice phase. The process of

34 us e d b e c a us e pr e vio us tr ia ls i n th e mod if ie d b loo d c oo le r s ho we d mo re da ma g e to Arg entine plants at that tempe rature than at -1C. When statistical analy sis of canopy leaf -dama g e ra tings we re not sig nificant for block (g rowth cha mber position) eff ects, a single controlled-f ree ze event wa s imposed on all the test lines. The EGC wa s programmed to maintain a -6 that temperature treatment from 2200 till 0800. Thirty bahiagrass lines were used to screen for LTFT. Vegetatively propagated lines were grown as previously described for the first LTFT screening experiment. Potted plants were enclosed in plastic bags to maintain humidity at a constant level. Relative humidity has been shown to shift the percent of leaf freeze-damage (Ivory and Whiteman, 1978) in freeze chamber trials with tropical grasses. The higher the relative humidity the more leaf freeze-damage was recorded at treatment temperatures ranging from -1 to -5C. Since potted plants came directly from a controlled greenhouse irrigation system, plastic bags were used to seal moisture and prevent desiccation of plants during the 10-h freeze-temperature treatment. Additionally, bagging individual pots prevented the compressor from failing due to ice formation when the large number of potted plants released moisture from the potting media as the fans recirculated the -6C air. Leaf vs. Root Effects Experiment 3 A third experiment was conducted using the modified blood cooler to determine if the observed freeze-damage was due to the entire plant (potted roots and leaves) being subject to low temperature or if the damage was only from the leaves being frozen. Potted plants of lines selected to represent a range of LTFT traits were immersed in controlled recirculation water baths (Polyscience Model 71, Niles IL) set at 5C. Potted plants were weighted down with river rock so the warmed recirculation water covered the

56 last to freeze (Pearce and Fuller, 2001b). If the xylem diameter is one of the mechanisms that is related to LTFT it might explain why older leaves freeze before younger leaves. Visualization of the third emerged fully expanded leaf cross-section of the freezetolerant (Figure 3-1) and freeze-sensitive (Figure 3-2) lines showed clear differences in xylem diameter as well as parenchyma cells within the midrib region. This was the region that was apparently damaged within hours after exposure to sunlight after a 10hour freeze. Figure 3-1.FL67 bahiagrass (freeze-tolerant) section showing bundle sheath, girder system of sclerenchymous tissue supporting the vascular bundle. Adaxial (bottom) midrib vascular bundle towards the left of center. Third emerged fully expanded leaf position at 100X. Figure 3-2.FL9 (freeze-sensitive) section showing larger vascular bundle area than FL67. Third emerged fully expanded leaf position at 100X.

65 of -2.27 MPa. This relationship ([(-2.27 MPa/-1.86C mol kg-1)/-2.27 MPa mol kg-1] = s/ f ) can be simplified, relating osmotic potential ( s) with freezing point-depression f : s = 0.537 f Figure 4-1 shows the relation between osmotic pressure (Pa), freezing temperature (C), osmolality (osmole kg-1), and relative humidity (%) of a solution. Figure 4-1.Relationship of osmolality, freezing temperature, osmotic pressure, and relative humidity of an aqueous solution with a pure solute (Source of figure from Wolf and Bryant, 2001). These relationships could be used to test the hypothesis that the mechanism of the LTFT trait is a result of freezing point-depression from solute increase in leaf symplast cells. Cells that would especially be important in the bahiagrass lamina would be those arranged radially in the bundle sheath adjacent to the xylem. This single layer of bundlesheath cells, which are immediately adjacent to the xylem vessel, is where the second photosynthetic carbon reduction occurs. The second layer of cells removed from the xylem vessels are mesophyll cells, where the primary photosynthetic carbon reduction reaction occurs. If ice forms initially in the xylem vessels, the single layer of bundle-

114 traditional g enetics, a s well as to furthe r investig ate the tr ait on a molecula r leve l. C o n f i r m a t i o n o f LT F T a s a t r a i t m a d e f u r t h e r e x p l o r a t i o n o f m e c h a n i s m s m e a n i n g f u l If L TF T m e c ha nis ms c ou ld b e ide nti fi e d, those mechanisms could be exploited to manipulate the trait. Another contribution for further scientific investigations is the observation that relative humidity needs to be controlled in freeze trials. The use of a plastic bag to enclose potted plants in a freeze trial may be beneficial in future experiments. Baffles and test plants, blocked by location, were used in the environmentally controlled chamber, where 30-lines were tested. A contribution to further investigations is the need to run small tests to reduce the CV and position effects in controlled freeze chamber experiments. A practical contribution of quantifying and confirming the LTFT trait in bahiagrass is the potential for plant breeders to use identified freeze-tolerant genotypes to extend the grazing season into the cool, fall period. Genotypes were identified and confirmed through controlled freezing trials, which withstood freeze events more severe than what is normally experienced in Florida. What was not achieved in the studies of LTFT trait range was solving the question of why an entire leaf would look damaged in a whorl of leaves, while the leaves adjacent to the completely damaged leaf appeared green. Canopy freeze-damage ratings were for the entire canopy of a potted plant. Undamaged leaves in a whorl tended to be those which had just emerged from the whorl through the first fully-expanded leaf. Canopy freeze-damage ratings were an average for the canopy leaves, and did not take into account the individual leaf freeze-damage behavior. Further investigations of bahiagrass leaf-tissue freeze-damage could record leaf damage by leaf position (and thus leaf age). Infrared thermography could be used to